Mixing in microfluidic structures is a challenging problem because in such structures the Reynolds number is characteristically very small (often much less than 1 and rarely greater than 200). At such low Reynolds numbers turbulent mixing does not occur and homogenization of solutions occurs by diffusion processes alone. While diffusional mixing of very small (and therefore rapidly diffusing species) can occur in a matter of seconds over distances of tens of micrometers, mixing of larger molecules such as peptides, proteins, high molecular weight nucleic acids can require equilibration times of many minutes to hours over comparable distances. Such delays are impractically long for many chemical analyses. This is particularly true in many microanalytical systems in which a desire for rapid throughput is a major impetus for their development.
Mixing speed may be increased if the two or more fluids to be mixed can be layered in a multitude of very thin alternating layers. This is true because the characteristic time for near equilibrium by diffusion (in the absence of gravitational sedimentation artifacts) is given as L2/D, where L is the distance between centers of adjacent fluid laminae, and D is the effective diffusivity of the slowest diffusion fluid constituent. Therefore, if the lamina thickness is decreased by a factor of 2 the mixing time decreases by a factor of 4. The effect associated with yet thinner laminae is obvious by extension. All active mixing devices operate on the principle of shredding and layering thinner and thinner laminae from macro- to meso- to microscale devices. This statement is true for devices that can induce turbulent flow as well. In turbulent mixing the shredding and layering of the lamina is random as are the fluid particle motions. Below are listed methods of active mixing with relevance to microfluidic mixing.
Ultrasonic/Piezoceramic Excitation
Ultrasonic plate waves created using piezoelectric films on silicon substrates have been used to generate recirculating flow patterns in reactor chambers (White 1996). This technique is also the subject of 3 U.S. patents (Northrup and White 1997). The use of piezoceramic excitation coupled to air and subsequently to a hundreds of picoliters stack of reagents has been demonstrated in glass capillaries (Evensen, Meldrum et al. 1998). In this method shear of the fluid near the wall significantly reduces the time required to achieve a homogeneous mixture. This device is fairly complex, requiring the addition of a transducer to the system. Excess ultrasound energy can damage components of the fluid.
Mixing Enhancement Using Passive Fluid Structures
Other researchers have attempted to create unique structures to achieve many fluid laminae using converging fluid flow profiles alone. One concept injects a multitude of microplumes of one reagent into another using a square 400 micronozzle in a 2 mm by 2 mm region (Elwenspoek, Lammerink et al. 1994). Another concept is to split and recombine fluid streams such that the lamina thickness is reduced each time the structure is reapplied (Krog, Branebjerg et al. 1996). These devices are difficult to manufacture. Mixing is also dependent on flowxe2x80x94in the absence of flow no mixing whatsoever occurs.
Electroxc3x6smotic Pumping
A few researchers have mixed one or more fluid streams using electroxc3x6smotic pumping as the means of delivering the fluid to a mixing junction (Manz, Effenhauser et al. 1994). However, this means of fluid delivery is not an active mixing configuration and only provides a means of delivering two fluids to a junction in a fashion similar to that which could be provided by any other pumping means.
All of the methods discussed above involve use of structures that are difficult to manufacture or require the presence (on or off the microfabricated device) of a bulky mechanical actuator. Some operate only when the fluid is flowing, and at a rate proportional to the fluid flow rate. What is needed is a generally applicable method for mixing arbitrarily small volumes of fluids that can be turned on and off at will, and that can be controlled by the user.
The present invention allows incorporation of a batch or continuous mixing capability into any meso- or microfluidic device by providing an electric field in a meso- or microfluidic channel. The electric field is generated by introducing two or more electrodes spaced by less than a few millimeters into a meso- or microfluidic channel to create a mixing region. Such electrodes may be made of any of several materials including gold. Electrodes may be plated or evaporated onto channel walls, or incorporated as separate pieces of metal, e.g., plates, wires or grids, into a channel made of nonconductive materials, such as polymers. The mixing region also contains chargeable surfaces that are substantially in contact with the electric field generated by at least some of the electrodes. These chargeable surfaces may be the walls of the channel, provided as a coating on those walls or provided as elements separate from the walls and appropriately positioned with respect to the electrodes. No alterations of the geometry of existing flow paths need be made, and the degree of mixing in the device can be controlled by the length of the electrodes, the flow rate past the electrodes, and the voltage applied to those electrodes. The degree of mixing can also be affected by choice of materials for the chargeable surface (in some cases by the selection of materials or coatings for channel walls) and the ionic strength of the fluids and the type and concentration of ions in the fluids. The method and device of this invention are preferably applied to fluids having low ionic strength less than or equal to about 1 mM. For example, electroosmotic mixing can be affected by varying the concentration of mono-, di-, tri- or tetravalent cations in the fluid (e.g., monovalent ions include K+ or Na+, divalent ions include Ca2+ or Mg2+, trivalent ions include Al3+ and tetravalent ions include Th4xe2x88x92).
By frustrating electroxc3x6smotic pumping by confining the fluid being pumped to a space that has closed ends in the direction of electroosmotic pumping, fluid is caused to recirculate within that space. We demonstrate that this electroosmotic recirculation of fluid, typically in the form of two contra-rotating vortices, is capable of rapidly mixing two or more fluids in that space, or of homogenizing a single fluid. When the distance or gap between two electrodes in a channel is less than a few millimeters, such mixing can occur within seconds and at voltages low enough to prevent formation of bubbles in the channel. The device can cause mixing in static fluids or in fluids flowing through a channel. In a specific embodiment, two electrodes form at least portions of two walls of the channel and the chargeable substrate is formed at least by portions of the remaining walls of the channel. In a rectangular shaped column of this embodiment, the axes of rotation of the vortices are parallel to the direction of flow in the channel. This mixer is applicable to aqueous and non-aqueous solutions, can be switched from xe2x80x9coffxe2x80x9d to mixing (i.e., to xe2x80x9conxe2x80x9d) at high rates with infinite gradations, has no moving parts and is extremely simple to manufacture. The ionic strength of the fluid or fluids to be mixed must be sufficiently low to allow electro{umlaut over (0)}smotic flow. The mixing device and methods of this invention provide a solution to the universal problem of mixing small volumes of fluids. They are ideally suited for use in microfluidic chemical analytical systems such as lab-on-a-chip applications.
More specifically, the invention provides meso- and microfluidic channels having an electroxc3x6smotic mixing region. One or more fluids carried in the channel or introduced into the channel can be mixed in this region. The mixing region of the channel comprises at least two electrodes which are separated from each other by an electrode gap (at most the width or depth of the channel). Voltage can be applied across these electrodes to generate an electric field in the channel. The mixing region of the channel also comprises at least two surfaces that can carry a surface charge, i.e. chargeable surfaces, when in contact with the fluid or fluids in the channel. The chargeable surfaces are positioned in the channel with respect to the electrodes such that electric field generated by at least two of the electrodes extends to the chargeable surfaces to cause electroosmotic flow. In specific embodiments the chargeable surfaces and electrodes extend about the same length and are coextensive with each other along the channel. In additional specific embodiments two electrodes are on opposite sides of the channel and two chargeable surfaces are on opposite sides of the channel and the chargeable surfaces are preferably substantially perpendicular to the electrode surfaces.
The meso- and microfluidic channels of this invention can be any regular shape, including among others rectangular, square, trapezoid or circular or any irregular shape. The mixing region can, for example, be constructed by positioning two electrodes within a tubular channel with the remaining curved sides of the tube serving as the chargeable surfaces.